Dataset Information

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

ABSTRACT: We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

SUBMITTER: Nam LB

PROVIDER: S-EPMC8455929 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

Nam Le Ba LB Choi Won Jun WJ Keum Young-Sam YS

Frontiers in pharmacology 20210908

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nucle ...[more]

PMID: 34566633

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Project description:Nuclear Nrf2 (nNrf2) binding to the antioxidant response element may promote chemoresistance in colorectal cancer. However, the shuttling of Nrf2 between cytoplasm and nucleus in colon cancer cells has revealed the possibility that cytoplasmic location of Nrf2 (cNrf2) may play a specific role in chemoresistance. Transfection of a nuclear location sequence (NLS)-wild-type or NLS-mutated Nrf2 expression vector into a stable shNrf2 HCT116 clone using the MTT assay to examine whether chemoresistance induced by cNrf2 may be greater than nNrf2. Different specific inhibitors and small hairpin (sh)RNAs of targeting genes were used to verify the mechanistic action of cNrf2 in chemoresistance and further confirmed by an animal model. The association of cNrf2 with chemotherapeutic response in patients with colorectal cancer was statistically analyzed. The MTT assay indicated that cNrf2 may play a more important role than nNrf2 in conferring 5-fluorouracil (5-FU) and oxaliplatin resistance in HCT116 cells. Mechanistically, cNrf2-induced PSMD4 expression was responsible for chemoresistance in the NLS-mutated Nrf2-tranfected shNrf2HCT116 clone via the NF-κB/AKT/β-catenin/ZEB1 cascades. The tumor burden induced by the NLS-mutated Nrf2-transfected shNrf2HCT116 clone was completely suppressed by treatment with 5-FU in combination with carfilzomib. A higher prevalence of unfavorable chemotherapeutic response in colorectal cancer patients with cNrf2, PSMD4-positive, p-p65-positive, and nuclear β-catenin tumors was observed when compared to their counterparts. cNrf2 may play a more important role than nNrf2 in the chemoresistance of colorectal cancer. Activation of the NF-κB/AKT/β-catenin/ZEB1 cascade by PSMD4 may be responsible for cNrf2-mediated chemoresistance.Condensed abstractCNrf2 may play a more important role than nNrf2 in conferring 5-FU and oxaliplatin resistance. This observation in patients seemed to support the findings of the cell and animal models and suggested that PSMD4 may be responsible cNrf2-mediated chemoresistance via the NF-κB/AKT/β-catenin /ZEB1 cascades.

Project description:BackgroundTriptolide (TP), a principal active substance from Tripterygium wilfordii, exhibits various pharmacological effects. However, its potential hepatotoxicity has always been a significant concern in clinical applications.PurposeThis research aimed to explore the involvement of ferroptosis in TP-mediated hepatic injury and the underlying mechanisms.MethodsIn this study, in vitro and in vivo experiments were involved. Hepatocyte damage caused by TP was evaluated using MTT assays, liver enzyme measurement and H&E staining technique. Ferroptosis was assessed by measuring iron level, lipid peroxide, glutathione (GSH), mitochondrial morphology and the key protein/mRNA expression implicated in ferroptosis. To verify the contribution of ferroptosis to TP-induced liver damage, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) and a plasmid for overexpressing glutathione peroxidase 4 (GPX4) were employed. Subsequently, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice and Nrf2 overexpression plasmid were utilized to investigate the underlying mechanisms. Nontargeted lipidomics was used to analyze lipid metabolism in mouse liver. Moreover, the cellular thermal shift assay (CETSA), cycloheximide (CHX) and MG132 treatments, and immunoprecipitation (IP) assays were applied to validate the binding of TP to Nrf2 and their interactions.ResultsTP triggered ferroptosis in hepatocytes, as indicated by iron accumulation and lipid peroxidation. Ferroptosis was responsible for TP-induced hepatic injury. During the process of TP-induced liver damage, the Nrf2 signaling pathway was significantly suppressed. Notably, the deletion of Nrf2 in mice aggravated the extent of liver injury and ferroptosis associated with TP, whereas enhancing Nrf2 expression in cells significantly reduced TP-induced ferroptosis. Additionally, dysregulation of lipid metabolism was associated with TP-induced liver injury. TP may directly bind to Nrf2 and enhance its degradation through the ubiquitin-proteasome pathway, thereby inhibiting or reducing Nrf2 expression.ConclusionIn summary, the suppression of Nrf2 by TP facilitated the occurrence of ferroptosis, resulting in liver damage.

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Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

Publications

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

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